基于内质网应激的牡荆素抗糖氧剥夺后恢复的细胞损伤作用及机制

The effect and mechanism of vitexin on the recovery of cells after glucose and oxygen deprivation based on endoplasmic reticulum stress

关键词:  牡荆素  糖氧剥夺后恢复  内质网应激  SH-SY5Y  PC12

Key words:  vitexin  recovery after glucose oxygen deprivation  endoplasmic reticulum stress  SH-SY5Y  PC12

摘要:

目的 探讨牡荆素通过内质网应激相关通路调控糖氧剥夺后恢复(oxygen glucose deprivation/re-oxygenation，OGD/R)SH-SY5Y细胞及PC12细胞炎症因子，从而阐明牡荆素对OGD/R损伤的保护作用机制。方法 建立OGD/R诱导SH-SY5Y细胞及PC12细胞模型，将其分为对照组(Control)、OGD/R、OGD/R+牡荆素(Vitexin)0.01μM、OGD/R+ Vitexin 0.1μM组及OGD/R+ Vitexin 1μM组，CCK-8法检测细胞活力的影响；流式细胞数检测活性氧(reactive oxygen species，ROS)水平；免疫荧光检测MBP；PCR检测内质网应激相关基因GRP78、CHOP的mRNA水平及炎症因子IL-6、TNF-α的表达的水平；Western blot检测内质网应激相关蛋白GRP78、ATF6、ATF4、CHOP。结果 Vitexin显著升高细胞活力(P＜0.05)，ROS水平显著降低(P＜0.01)，MBP蛋白表达水平显著升高(P＜0.01)；抑制内质网应激相关蛋白GRP78、ATF6、ATF4、CHOP表达(P＜0.01)，内质网应激相关基因GRP78、CHOP的mRNA水平显著降低；降低细胞炎症因子IL-6、TNF-α(P＜0.01)。结论 在SH-SY5Y细胞及PC12细胞中，牡荆素有效剂量分别为1μM及0.01-1μM，有效抑制ROS产生，促进MBP表达，缓解受损神经细胞内质网应激导致的炎症损伤，发挥一定的神经保护作用。

Abstract:

OBJECTIVE To investigate the regulation of inflammatory factors of OGD/R SH-SY5Y cells and PC12 cells by endoplasmic reticulum stress-related pathways, and to clarify the protective mechanism of vitexin against OGD/R injury. METHODS OGD/R-induced SH-SY5Y cell and PC12 cell models were established. They were divided into Control group (Control group), model group (OGD/R), OGD/R+ Vitexin (Vitexin) 0.01μM group, OGD/R+ Vitexin 0.1μM group and OGD/R+ Vitexin 1μM group. CCK-8 assay was performed to evaluate the impact on cell viability. Flow cytometry was used to measure the level of reactive oxygen species (ROS). Immunofluorescence was used to detect the expression of myelin basic protein (MBP). PCR was performed to measure the mRNA levels of endoplasmic reticulum stress-related genes GRP78 and CHOP, as well as the expression levels of inflammatory factors IL-6 and TNF-α. Western blot was conducted to detect the expression of endoplasmic reticulum stress-related proteins GRP78, ATF6, ATF4, and CHOP. RESULTS Vitexin significantly increased cell viability (P<0.05), reduced ROS levels (P<0.01), and increased the expression level of MBP protein (P<0.01). It inhibited the expression of endoplasmic reticulum stress-related proteins GRP78, ATF6, ATF4, and CHOP (P<0.01), and decreased the mRNA levels of endoplasmic reticulum stress-related genes GRP78 and CHOP. It also reduced the levels of inflammatory cytokines IL-6 and TNF-α (P<0.01). CONCLUSION In SH-SY5Y cells and PC12 cells, the effective dose of vitexin was 1μM and 0.01-1μM, respectively, which effectively inhibited ROS production, promoted MBP expression, alleviated the inflammatory injury caused by endoplasmic reticulum stress of damaged nerve cells, and played a certain neuroprotective role.